Producing potent high-affinity antibodies that can neutralize target pathogens remains a central goal for vaccine research. While a great deal is known about antibody molecules themselves, still too little is understood about the programming of high-affinity memory B cells that produce them. Many promising vaccine antigens fail to elicit protective antibodies using contemporary vaccine formulations. Thus, major gaps remain in our basic understanding of antibody affinity maturation and how to enhance poor immunogenicity using rational protein vaccination. Research Focus: GCs are dynamic microenvironments that emerge within secondary lymphoid tissues following exposure to foreign antigens. The evolution of antibody specificity within these GC reactions is required to generate high-affinity memory B cells and long-lived plasma cells. Follicular helper T (TFH) cells provide the molecular cues for initiation of GCs and control of ongoing evolutionary mechanisms within the GC reaction. Understanding the regulatory mechanisms that control memory BCR evolution and GC B cell function remain an important basic goal for the field and is the major focus of the current application. Specific Aims: We have developed multi-dimensional single cell strategies to track these complex cellular behaviors at the clonal and sub-clonal levels in vivo. We will use this approach: SA-1: to study the molecular control of secondary GC induction and function at recall SA-2: to dissect the stage-specific cyclic progression of GC transcriptional programming SA-3: to modify the molecular control of GC B cell evolution and GC exit. Impact: We use polyclonal murine models of protein vaccination with state-of-the-art single cell analyses of antigen-specific immune function. These high-resolution studies of model antigens have the capacity to shift the basic conceptual framework that surrounds existing vaccine paradigms. Importantly, these basic new principles and assays can be used to re-design contemporary vaccine formulations that optimize high-affinity B cell immunity to more complex antigens.